(1) Statement of the Invention
The present invention relates to chemiluminescent 1,2-dioxetane compounds which can be triggered by an activating agent to generate light. In particular, the present invention relates to stable aryl group substituted 1,2-dioxetanes which contain an activatable oxide group (OX) which is ring substituted in the aryl group, wherein the stable 1,2-dioxetane forms an unstable 1,2-dioxetane compound by removal of X which decomposes to light and two carbonyl containing compounds.
(2) Prior Art
1. Mechanisms of Luminescence. Exothermic chemical reactions release energy during the course of the reaction. In virtually all cases, this energy is in the form of vibrational excitation or heat. However, a few chemical processes generate light or chemiluminescence instead of heat. The mechanism for light production involves two steps: (1) thermal or catalyzed decomposition of a high energy material (generally a peroxide) yields one of the reaction products in a triplet or singlet electronic excited state and (2) emission of a photon (fluorescence or phosphorescence) from this excited species produces the light observed from the reaction. ##STR2##
2. Dioxetane Intermediates in Bioluminescence. In 1968 McCapra proposed that 1,2-dioxetanes might be the key high-energy intermediates in various bioluminescent reactions including the firefly system. (F. McCapra. Chem. Commun., 155 (1968)). Although this unstable dioxetane intermediate has not been isolated nor observed spectroscopically, unambiguous evidence for its intermediacy in this biochemical reaction has been provided by oxygen-18 labelling experiments. (O. Shimomura and F. H. Johnson, Photochem. Photobiol., 30, 89 (1979)). ##STR3##
3. First Synthesis of Authentic 1,2-Dioxetanes. In 1969 Kopecky and Mumford reported the first synthesis of a dioxetane (3,3,4-trimethyl-1,2-dioxetane) by the base-catalyzed cyclization of a beta-bromohydroperoxide. (K. R. Kopecky and C. Mumford, Can. J. Chem., 47, 709 (1969)). As predicted by McCapra, this dioxetane did, in fact, produce chemiluminescence upon heating to 50.degree. C. with decomposition to acetone and acetaldehyde. However, this peroxide is relatively unstable and cannot be stored at room temperature (25.degree. C.) without rapid decomposition. In addition, the chemiluminescence efficiency is very low (less than 0.1%). This inefficiency is due to two factors: (1) the biradical nature of the mechanism for its decomposition and (2) the low quantum yield of fluorescence of the carbonyl cleavage products. ##STR4##
Bartlett and Schaap and Mazur and Foote independently developed an alternate and more convenient synthetic route to 1,2-dioxetanes. Photooxygenation of properly-substituted alkenes in the presence of molecular oxygen and a photosensitizing dye produces the dioxetanes in high yields. (P. D. Bartlett and A. P. Schaap, J. Amer. Chem. Soc., 92, 3223 (1970) and S. Mazur and C. S. Foote, J. Amer. Chem. Soc., 92 3225 (1970)). The mechanism of this reaction involves the photochemical generation of a metastable species known as singlet oxygen which undergoes 2+2 cycloaddition with the alkene to give the dioxetane. Research has shown that a variety of dioxetanes can be prepared using this reaction (A. P. Schaap, P. A. Burns, and K. A. Zaklika, J. Amer. Chem. Soc., 99, 1270 (1977); K. A. Zaklika, P. A. Burns, and A. P. Schaap, J. Amer. Chem. Soc., 100, 318 (1978); K. A. Zaklika, A. L. Thayer, and A. P. Schaap, J. Amer. Chem. Soc., 100, 4916 (1978); K. A. Zaklika, T. Kissel, A. L. Thayer, P. A. Burns, and A. P. Schaap, Photochem. Photobiol., 30, 35 (1979); and A. P. Schaap, A. L. Thayer, and K. Kees, Organic Photochemical Synthesis, II, 49 (1976)). During the course of this research, a polymer-bound sensitizer for ##STR5## photooxygenations was developed (A. P. Schaap, A. L. Thayer, E. C. Blossey, and D. C. Neckers, J. Amer. Chem. Soc., 97, 3741 (1975); and A. P. Schaap, A. L. Thayer, K. A. Zaklika, and P. C. Valenti, J. Amer. Chem. Soc., 101, 4016 (1979)). This new type of sensitizer has been patented and sold under the tradename SENSITOX.TM. (U.S. Pat. No. 4,315,998 (2/16/82); Canadian Pat. No. 1,044,639 (12/19/79)). Over fifty references have appeared in the literature reporting the use of this product.
4. Preparation of Stable dioxetanes Derived from Sterically Hindered Alkenes. Wynberg discovered that photooxygenation of sterically hindered alkenes such as adamantylideneadamantane affords a very stable dioxetane (J. H. Wieringa, J. Strating, H. Wynberg, and W. Adam, Tetrahedron Lett., 169 (1972)). A collaborative study by Turro and Schaap showed that this dioxetane exhibits an activation energy for decomposition of 37 kcal/mol and a half-life at room ##STR6## temperature (25.degree. C.) of over 20 years (N. J. Turro, G. Schuster, H. C. Steinmetzer, G. R. Faler and A. P. Schaap, J. Amer. Chem. Soc., 97, 7110 (1975)). In fact, this is the most stable dioxetane yet reported in the literature. Adam and Wynberg have recently suggested that functionalized adamantylideneadamantane 1,2-dioxetanes may be useful for biomedical applications (W. Adam, C. Babatsikos, and G. Cilento, Z. Naturforsch., 39b, 679 (1984); H. Wynberg, E. W. Meijer, and J. C. Hummelen, In Bioluminescence and Chemiluminescence, M. A. DeLuca and W. D. McElroy (Eds.). Academic Press, New york, p. 687, 1981). However, use of this extraordinarily stable peroxide for chemiluminescent labels would require detection temperatures of 150.degree. to 250.degree. C. Clearly, these conditions are unsuitable for the evaluation of biological analytes in aqueous media. Further, the products (adamantanones) of these dioxetanes are only weakly fluorescent so that the chemiluminescent decomposition of these proposed inefficient. McCapra, Adam, and Foote have shown that incorporation of a spirofused cyclic or polycyclic alkyl group with a dioxetane can help to stabilize dioxetanes that are relatively unstable in the absence of this sterically bulky group (F. McCapra, I. Beheshti, A. Burford, R. A. Hann, and K. A. Zaklika, J. Chem. Soc., Chem. Commun., 944 (1977); W. Adam, L. A. A. Encarnacion, and K. Zinner, Chem. Ber., 116, 839 (1983); and G. G. Geller, C. S. Foote, and D. B. Pechman, Tetrahedron Lett., 673 (1983)).
5. Effects of Substituents on Dioxetane Chemiluminescence. The stability and the chemiluminescence efficiency of dioxetanes can be altered by the attachment of specific substituents to the peroxide ring (K. A. Zaklika, T. Kissel, A. L. Thayer, P. A. Burns, and A. P. Schaap, Photochem. Photobiol., 30, 35 (1979); A. P. Schaap and S. Gagnon, J. Amer. Chem. Soc., 104, 3504 (1982); A. P. Schaap, S. Gagnon, and K. A. Zaklika, Tetrahedron Lett., 2943 (1982); and R. S. Handley, A. J. Stern, and A. P. Schaap, Tetrahedron Lett., 3183 (1985)). The results with the bicyclic system shown below illustrate the profound effect of various functional groups on the properties of dioxetanes. The hydroxy-substituted dioxetane (X.dbd.OH) derived from the 2,3-diaryl-1,4-dioxene exhibits a half-life for decomposition at room temperature ##STR7## (25.degree. C.) of 57 hours and produces very low levels of luminescence upon heating at elevated temperatures. In contrast, however, reaction of this dioxetane with a base at -30/.degree. C. affords a flash of blue light. Kinetic studies have shown that the deprotonated dioxetane (X.dbd.O.sup.-) decomposes 5.7.times.10.sup.6 times faster than the protonated form (X.dbd.OH) at 25.degree. C.
The differences in the properties of these two dioxetanes arise because of two competing mechanisms for decomposition (K. A. Zaklika, T. Kissel, A. L. Thayer, P. A. Burns, and A. P. Schaap, Photochem. Photobiol., 30, 35 (1979); A. P. Schaap and S. Gagnon, J. Amer. Chem. Soc., 104 3504 (1982); A. P. Schaap, S. Gagnon, and K. A. Zaklika, Tetrahedron Lett., 2943 (1982); and R. S. Handley, A. J. Stern, and A. P. Schaap, Tetrahedron Lett., 3183 (1985). Stable dioxetanes cleave by a process that requires approximately 25 kcal for homolysis of the O--O bond and formation of a biradical. An alternative mechanism for decomposition is available to dioxetanes bearing substituents such as O.sup.- with low oxidation potentials. The cleavage is initiated by intramolecular electron transfer from the substituent to the antibonding orbital of the peroxide bond. In contrast to the biradical mechanism, the electron-transfer process generates chemiluminescence with high efficiency.